EP2512225A1 - Use of vip3ab in combination with cry1ca for management of resistant insects - Google Patents
Use of vip3ab in combination with cry1ca for management of resistant insectsInfo
- Publication number
- EP2512225A1 EP2512225A1 EP10842622A EP10842622A EP2512225A1 EP 2512225 A1 EP2512225 A1 EP 2512225A1 EP 10842622 A EP10842622 A EP 10842622A EP 10842622 A EP10842622 A EP 10842622A EP 2512225 A1 EP2512225 A1 EP 2512225A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- plants
- plant
- protein
- seeds
- refuge
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000238631 Hexapoda Species 0.000 title claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 189
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 134
- 230000000749 insecticidal effect Effects 0.000 claims abstract description 38
- 238000000034 method Methods 0.000 claims abstract description 30
- 241000256251 Spodoptera frugiperda Species 0.000 claims abstract description 14
- 238000011161 development Methods 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims description 88
- 240000008042 Zea mays Species 0.000 claims description 24
- 239000000203 mixture Substances 0.000 claims description 24
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 18
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 17
- 235000005822 corn Nutrition 0.000 claims description 17
- 230000009261 transgenic effect Effects 0.000 claims description 11
- 229920000742 Cotton Polymers 0.000 claims description 7
- 244000068988 Glycine max Species 0.000 claims description 5
- 235000010469 Glycine max Nutrition 0.000 claims description 4
- ZHVOBYWXERUHMN-KVJKMEBSSA-N 3-[(3s,5r,8r,9s,10s,13s,14s,17s)-10,13-dimethyl-3-[(2r,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2h-furan-5-one Chemical compound O([C@@H]1C[C@H]2CC[C@@H]3[C@@H]([C@]2(CC1)C)CC[C@]1([C@H]3CC[C@@H]1C=1COC(=O)C=1)C)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O ZHVOBYWXERUHMN-KVJKMEBSSA-N 0.000 claims description 3
- 244000038559 crop plants Species 0.000 claims 5
- 241000219146 Gossypium Species 0.000 claims 1
- 108700012359 toxins Proteins 0.000 description 121
- 235000018102 proteins Nutrition 0.000 description 114
- 239000003053 toxin Substances 0.000 description 95
- 231100000765 toxin Toxicity 0.000 description 94
- 210000004027 cell Anatomy 0.000 description 38
- 108091028043 Nucleic acid sequence Proteins 0.000 description 26
- 241000607479 Yersinia pestis Species 0.000 description 26
- 230000009471 action Effects 0.000 description 21
- 241001147398 Ostrinia nubilalis Species 0.000 description 16
- 230000027455 binding Effects 0.000 description 16
- 235000001014 amino acid Nutrition 0.000 description 15
- 235000021028 berry Nutrition 0.000 description 14
- 239000000575 pesticide Substances 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 12
- 108020003175 receptors Proteins 0.000 description 11
- 102000005962 receptors Human genes 0.000 description 11
- 239000013598 vector Substances 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 238000009472 formulation Methods 0.000 description 10
- 230000009466 transformation Effects 0.000 description 10
- 108020004414 DNA Proteins 0.000 description 9
- 244000005700 microbiome Species 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 241000724266 Broad bean mottle virus Species 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 230000000361 pesticidal effect Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 238000006467 substitution reaction Methods 0.000 description 8
- 206010034133 Pathogen resistance Diseases 0.000 description 7
- 230000000306 recurrent effect Effects 0.000 description 7
- 241000894006 Bacteria Species 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- AYDAHOIUHVUJHQ-UHFFFAOYSA-N 1-(3',6'-dihydroxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-5-yl)pyrrole-2,5-dione Chemical compound C=1C(O)=CC=C2C=1OC1=CC(O)=CC=C1C2(C1=CC=2)OC(=O)C1=CC=2N1C(=O)C=CC1=O AYDAHOIUHVUJHQ-UHFFFAOYSA-N 0.000 description 5
- 241000193388 Bacillus thuringiensis Species 0.000 description 5
- 229940097012 bacillus thuringiensis Drugs 0.000 description 5
- 230000004071 biological effect Effects 0.000 description 5
- 239000002596 immunotoxin Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 239000008188 pellet Substances 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 230000007704 transition Effects 0.000 description 5
- 239000008001 CAPS buffer Substances 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 208000037065 Subacute sclerosing leukoencephalitis Diseases 0.000 description 4
- 206010042297 Subacute sclerosing panencephalitis Diseases 0.000 description 4
- 125000003275 alpha amino acid group Chemical group 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000006192 iodination reaction Methods 0.000 description 4
- 239000003550 marker Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 231100000654 protein toxin Toxicity 0.000 description 4
- 239000002689 soil Substances 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 108700003918 Bacillus Thuringiensis insecticidal crystal Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 241000233866 Fungi Species 0.000 description 3
- 241001147381 Helicoverpa armigera Species 0.000 description 3
- 241001310335 Paenibacillus lentimorbus Species 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 230000002860 competitive effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 231100000673 dose–response relationship Toxicity 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 210000003000 inclusion body Anatomy 0.000 description 3
- 239000002917 insecticide Substances 0.000 description 3
- 230000026045 iodination Effects 0.000 description 3
- 230000001418 larval effect Effects 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000003094 microcapsule Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 230000002588 toxic effect Effects 0.000 description 3
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 3
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 2
- 241000588986 Alcaligenes Species 0.000 description 2
- 241000223651 Aureobasidium Species 0.000 description 2
- 241000589151 Azotobacter Species 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 241000122106 Diatraea saccharalis Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 108010042653 IgA receptor Proteins 0.000 description 2
- 241000235649 Kluyveromyces Species 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- 241000721703 Lymantria dispar Species 0.000 description 2
- 241000215495 Massilia timonae Species 0.000 description 2
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 241000589540 Pseudomonas fluorescens Species 0.000 description 2
- 241000589180 Rhizobium Species 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000003139 biocide Substances 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- -1 dditional Species 0.000 description 2
- 239000002270 dispersing agent Substances 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 239000000834 fixative Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000012669 liquid formulation Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000000329 molecular dynamics simulation Methods 0.000 description 2
- 230000009871 nonspecific binding Effects 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000002285 radioactive effect Effects 0.000 description 2
- 238000000163 radioactive labelling Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000589220 Acetobacter Species 0.000 description 1
- 244000235858 Acetobacter xylinum Species 0.000 description 1
- 235000002837 Acetobacter xylinum Nutrition 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 241000186063 Arthrobacter Species 0.000 description 1
- 241000212384 Bifora Species 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 239000011547 Bouin solution Substances 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000197194 Bulla Species 0.000 description 1
- 101150078024 CRY2 gene Proteins 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 239000003298 DNA probe Substances 0.000 description 1
- 241001631715 Dendrolimus Species 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 241001057636 Dracaena deremensis Species 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241000190714 Gymnosporangium clavipes Species 0.000 description 1
- 241000255967 Helicoverpa zea Species 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- 241000588748 Klebsiella Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000255777 Lepidoptera Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001443590 Naganishia albida Species 0.000 description 1
- 241000033319 Naganishia diffluens Species 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000346285 Ostrinia furnacalis Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001310339 Paenibacillus popilliae Species 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 241000222051 Papiliotrema laurentii Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 241000500437 Plutella xylostella Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 241000589615 Pseudomonas syringae Species 0.000 description 1
- 241000158450 Rhodobacter sp. KYW73 Species 0.000 description 1
- 241000191043 Rhodobacter sphaeroides Species 0.000 description 1
- 241000190932 Rhodopseudomonas Species 0.000 description 1
- 241000223252 Rhodotorula Species 0.000 description 1
- 241000223253 Rhodotorula glutinis Species 0.000 description 1
- 241000223254 Rhodotorula mucilaginosa Species 0.000 description 1
- 241000235070 Saccharomyces Species 0.000 description 1
- 241001479507 Senecio odorus Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- 241000607715 Serratia marcescens Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000222068 Sporobolomyces <Sporidiobolaceae> Species 0.000 description 1
- 241000123675 Sporobolomyces roseus Species 0.000 description 1
- 241000187747 Streptomyces Species 0.000 description 1
- 244000288561 Torulaspora delbrueckii Species 0.000 description 1
- 235000014681 Torulaspora delbrueckii Nutrition 0.000 description 1
- 241001495125 Torulaspora pretoriensis Species 0.000 description 1
- 241000589634 Xanthomonas Species 0.000 description 1
- 241000589636 Xanthomonas campestris Species 0.000 description 1
- 241000209149 Zea Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 230000002924 anti-infective effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229960005475 antiinfective agent Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- OCBHHZMJRVXXQK-UHFFFAOYSA-M benzyl-dimethyl-tetradecylazanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 OCBHHZMJRVXXQK-UHFFFAOYSA-M 0.000 description 1
- GINJFDRNADDBIN-FXQIFTODSA-N bilanafos Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCP(C)(O)=O GINJFDRNADDBIN-FXQIFTODSA-N 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000000853 biopesticidal effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 210000003850 cellular structure Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000031902 chemoattractant activity Effects 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000010410 dusting Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000004495 emulsifiable concentrate Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229910052736 halogen Inorganic materials 0.000 description 1
- 230000002140 halogenating effect Effects 0.000 description 1
- 150000002367 halogens Chemical class 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000014666 liquid concentrate Nutrition 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 231100001225 mammalian toxicity Toxicity 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 210000000110 microvilli Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 238000003322 phosphorimaging Methods 0.000 description 1
- 229910052615 phyllosilicate Inorganic materials 0.000 description 1
- 230000019612 pigmentation Effects 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000010152 pollination Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001525 receptor binding assay Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000007888 toxin activity Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000004563 wettable powder Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/40—Fabaceae, e.g. beans or peas
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/50—Cotton
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- Bt proteins have been used to create the insect-resistant transgenic plants that have been successfully registered and commercialized to date. These include CrylAb, CrylAc, CrylF and Cry3Bb in corn, CrylAc and Cry2Ab in cotton, and Cry3A in potato.
- the commercial products expressing these proteins express a single protein except in cases where the combined insecticidal spectrum of 2 proteins is desired (e.g., CrylAb and Cry3Bb in corn combined to provide resistance to lepidopteran pests and rootworm, respectively) or where the independent action of the proteins makes them useful as a tool for delaying the development of resistance in susceptible insect populations (e.g., CrylAc and Cry2Ab in cotton combined to provide resistance management for tobacco budworm). See also U.S. Patent Application Publication No. 2009/0313717, which relates to a Cry2 protein plus a Vip3Aa, CrylF, or CrylA for control of Helicoverpa zea or armigerain.
- WO 2009/132850 relates to CrylF or CrylA and Vip3Aa for controlling Spodoptera frugiperda.
- U.S. Patent Application Publication No. 2008/031 1096 relates in part to CrylAb for controlling CrylF-resistant ECB.
- the proteins selected for use in an insect resistant management (IRM) stack need to exert their insecticidal effect independently so that resistance developed to one protein does not confer resistance to the second protein (i.e., there is not cross resistance to the proteins). If, for example, a pest population that is resistant to "Protein A” is sensitive to "Protein B", one would conclude that there is not cross resistance and that a combination of Protein A and Protein B would be effective in delaying resistance to Protein A alone.
- IRM insect resistant management
- CrylFa protein is useful in controlling many lepidopteran pests species including the European corn borer (ECB; Ostrinia nubilalis (Hubner)) and the fall armyworm (FAW; Spodoptera frugiperda), and is active against the sugarcane borer (SCB; Diatraea saccharalis) .
- the CrylFa protein as produced in transgenic corn plants containing event TCI 507, is responsible for an industry-leading insect resistance trait for FAW control.
- CrylFa is further deployed in the Herculex ® , Smarts taxTM, and
- Cry toxins are listed at the website of the official B. t. nomenclature committee (Crickmore et al.; lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).
- Cry Cry
- the subject invention relates in part to the use of a Vip3Ab protein in combination with a CrylCa protein. Plants (and acreage planted with such plants) that produce both of these proteins are included within the scope of the subject invention.
- the subject invention relates in part to the surprising discovery that Vip3Ab does not compete with CrylCa for binding sites in the gut of fall army worm (Spodoptera frugiperda; FAW).
- the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Vip3Ab and CrylCa being the base pair.
- the combination of the selected toxins provides non-cross-resistant action against FAW.
- Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus CrylFa, CrylDa, CrylBe, or CrylE as the third protein for targeting FAW.
- Additional toxins/genes can also be added according to the subject invention.
- CrylFa or CrylBe are stacked with the subject pair of proteins (both CrylFa and CrylBe are both active against both FAW and European cornborer (ECB))
- adding two additional proteins to this triple stack wherein the two added proteins target ECB would provide three sites of action against FAW, and three sites of action against ECB.
- These two added proteins could be selected from the group consisting of Cry2A, Cryll, DIG-3, and CrylAb. This would result in a five-protein stack having three sites of action against two insects (ECB and FAW).
- the subject invention relates in part to the surprising discovery that Vip3Ab and CrylCa do not compete for binding with each other in the gut of fall army worms (FAW; Spodoptera frugiperda).
- a Vip3Ab protein can be used in combination with a CrylCa protein in transgenic corn (and other plants; e.g., cotton and soybeans, for example) to delay or prevent FAW from developing resistance to either of these proteins alone.
- the subject pair of proteins can be effective at protecting plants (such as maize plants and/or soybean plants) from damage by Cry-resistant fall army worm. That is, one use of the subject invention is to protect corn and other economically important plant species from damage and yield loss caused by fall armyworm populations that could develop resistance to Vip3Ab or CrylCa.
- the subject invention thus teaches an insect resistant management (IRM) stack comprising Vip3Ab and CrylCa to prevent or mitigate the development of resistance by FAW to either or both of these proteins.
- IRM insect resistant management
- compositions for controlling lepidopteran pests comprising cells that produce a Vip3Ab insecticidal protein and a CrylCa insecticidal protein.
- the invention further comprises a host transformed to produce both a Vip3Ab insecticidal protein and a CrylCa insecticidal protein, wherein said host is a microorganism or a plant cell.
- the subject polynucleotide(s) are preferably in a genetic construct under control of a non-Bacillus-thuringiensis promoter(s).
- the subject polynucleotides can comprise codon usage for enhanced expression in a plant.
- the invention provides a method of controlling lepidopteran pests comprising contacting said pests or the environment of said pests with an effective amount of a composition that contains a Vip3 Ab core toxin-containing protein and further contains a CrylCa core toxin-containing protein.
- An embodiment of the invention comprises a maize plant comprising a plant- expressible gene encoding a CrylCa insecticidal protein and a plant-expressible gene encoding a Vip3 Ab insecticidal protein, and seed of such a plant.
- a further embodiment of the invention comprises a maize plant wherein a plant- expressible gene encoding a CrylCa insecticidal protein and a plant-expressible gene encoding a Vip3Ab insecticidal protein have been introgressed into said maize plant, and seed of such a plant.
- the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins, with Vip3Ab and CrylCa being the base pair.
- the selected toxins have three separate sites of action against FAW.
- Some preferred "three sites of action" pyramid combinations include the subject base pair of proteins plus CrylFa, CrylDa, Cry 1 Be, or Cry IE as the third protein for targetting FAW.
- synthetic sites of action it is meant any of the given proteins do not cause cross-resistance with each other.
- the subject pairs of toxins CrylFa plus Vip3Ab and CrylFa plus CrylCa provide non-cross-resistant action against FAW.
- the inability of Vip3Abl to compete for the binding of CrylCa in the gut of FAW demonstrates that these three protein toxins (CrylFa, Vip3Ab, and CrylCa) represent a triple-stack pyramid of Cry toxins that provide three separate target site interactions within the gut of FAW.
- These particular triple stacks would, according to the subject invention, advantageously and surprisingly provide non- cross-resistant action against FAW.
- these three proteins do not compete with each other, one skilled in the art will recognize that this can help to reduce or eliminate the requirement for refuge acreage.
- plants expressing the triple combination of CrylFa, Vip3Ab and CrylCa will be useful in delaying or preventing the development of resistance in FAW to the individual or combination of these proteins.
- Additional toxins/genes can also be added according to the subject invention. For example, if CrylFa or CrylBe are stacked with the subject pair of proteins (both CrylFa and CrylBe are both active against both FAW and European cornborer (ECB)), adding two additional proteins to this triple stack wherein the two added proteins target ECB, would provide three sites of action against FAW, and three sites of action against ECB.
- the fourth and fifth proteins could be selected from the group consisting of Cry2A, Cry II, DIG-3 (see U.S. Patent Application Serial No. 61/284,278 (filed December 16, 2009) and US 2010 00269223), and CrylAb. This would result in a five-protein stack having three sites of action against two insects (ECB and FAW)
- one deployment option is to use the subject pair of proteins in combination with a third toxin/gene, and to use this triple stack to mitigate the development of resistance in FAW to any of these toxins.
- the subject invention also relates in part to triple stacks or "pyramids" of three (or more) toxins.
- the selected toxins have three separate sites of action against FAW.
- Vip3Ab plus CrylCa plus CrylFa would, according to the subject invention, advantageously and surprisingly provide three sites of action against FAW. This can help to reduce or eliminate the requirement for refuge acreage.
- CrylFa is deployed in the Herculex ® , SmartStaxTM, and Wides StrikeTM products.
- the subject pair of genes (Vip3Ab and CrylCa) could be combined into, for example, a CrylFa product such as Herculex ® , SmartStaxTM, and WideStrikeTM. Accordingly, the subject pair of proteins could be significant in reducing the selection pressure on these andother proteins. The subject pair of proteins could thus be used as in the three gene combinations for corn and other plants (cotton and soybeans, for example).
- Plants (and acreage planted with such plants) that produce any of the subject combinations of proteins are included within the scope of the subject invention. Additional toxins/genes can also be added, but the particular stacks discussed above advantageously and surprisingly provide multiple sites of action against FAW and/or ECB. This can help to reduce or eliminate the requirement for refuge acreage. A field thus planted of over ten acres is thus included within the subject invention.
- GENBANK can also be used to obtain the sequences for any of the genes and proteins disclosed or mentioned herein. See Appendix A, below. Relevant sequences are also available in patents. For example, U.S. Patent No. 5,188,960 and U.S. Patent No. 5,827,514 describe CrylFa core toxin containing proteins suitable for use in carrying out the present invention. U.S. Patent No. 6,218,188 describes plant-optimized DNA sequences encoding CrylFa core toxin-containing proteins that are suitable for use in the present invention. USSN 61/284,275 (filed December 16, 2009) provides some truncated Cryl Ca proteins that can be used according to the subject invention.
- Combinations of proteins described herein can be used to control lepidopteran pests.
- Adult lepidopterans for example, butterflies and moths, primarily feed on flower nectar and are a significant effector of pollination.
- Nearly all lepidopteran larvae i.e., caterpillars, feed on plants, and many are serious pests.
- Caterpillars feed on or inside foliage or on the roots or stem of a plant, depriving the plant of nutrients and often destroying the plant's physical support structure.
- caterpillars feed on fruit, fabrics, and stored grains and flours, ruining these products for sale or severely diminishing their value.
- reference to lepidopteran pests refers to various life stages of the pest, including larval stages.
- Some chimeric toxins of the subject invention comprise a full N-terminal core toxin portion of a Bt toxin and, at some point past the end of the core toxin portion, the protein has a transition to a heterologous protoxin sequence.
- the N-terminal, insecticidally active, toxin portion of a Bt toxin is referred to as the "core" toxin.
- the transition from the core toxin segment to the heterologous protoxin segment can occur at approximately the toxin/protoxin junction or, in the alternative, a portion of the native protoxin (extending past the core toxin portion) can be retained, with the transition to the heterologous protoxin portion occurring downstream.
- one chimeric toxin of the subject invention is a full core toxin portion of CrylCa (roughly the first 600 amino acids) and/or a heterologous protoxin (the remaining amino acids to the C-terminus).
- the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin.
- the portion of a chimeric toxin comprising the protoxin is derived from a CrylAb protein toxin.
- Bt toxins even within a certain class such as CrylCa, will vary to some extent in length and the precise location of the transition from core toxin portion to protoxin portion.
- the CrylCa toxins are about 1150 to about 1200 amino acids in length.
- the transition from core toxin portion to protoxin portion will typically occur at between about 50% to about 60% of the full length toxin.
- the chimeric toxin of the subject invention will include the full expanse of this N-terminal core toxin portion.
- the chimeric toxin will comprise at least about 50% of the full length of the Cryl Bt toxin protein. This will typically be at least about 590 amino acids.
- the full expanse of the CrylAb protoxin portion extends from the end of the core toxin portion to the C-terminus of the molecule.
- genes and toxins useful according to the subject invention include not only the full length sequences disclosed but also fragments of these sequences, variants, mutants, and fusion proteins which retain the characteristic pesticidal activity of the toxins specifically exemplified herein.
- variants or mutants
- variants of genes refer to nucleotide sequences which encode the same toxins or which encode equivalent toxins having pesticidal activity.
- equivalent toxins refers to toxins having the same or essentially the same biological activity against the target pests as the claimed toxins.
- genes encoding active toxins can be identified and obtained through several means.
- the specific genes or gene portions exemplified herein may be obtained from the isolates deposited at a culture depository. These genes, or portions or variants thereof, may also be constructed synthetically, for example, by use of a gene synthesizer. Variations of genes may be readily constructed using standard techniques for making point mutations. Also, fragments of these genes can be made using commercially available exonucleases or endonucleases according to standard procedures. For example, enzymes such as Bal31 or site-directed mutagenesis can be used to systematically cut off nucleotides from the ends of these genes. Genes that encode active fragments may also be obtained using a variety of restriction enzymes. Proteases may be used to directly obtain active fragments of these protein toxins.
- a further method for identifying the genes encoding the toxins and gene portions useful according to the subject invention is through the use of oligonucleotide probes. These probes are detectable nucleotide sequences. These sequences may be detectable by virtue of an appropriate label or may be made inherently fluorescent as described in International Application No. WO93/16094. As is well known in the art, if the probe molecule and nucleic acid sample hybridize by forming a strong bond between the two molecules, it can be reasonably assumed that the probe and sample have substantial homology. Preferably, hybridization is conducted under stringent conditions by techniques well-known in the art, as described, for example, in Keller, G. FL, M. M.
- variant toxins Certain toxins of the subject invention have been specifically exemplified herein. Since these toxins are merely exemplary of the toxins of the subject invention, it should be readily apparent that the subject invention comprises variant or equivalent toxins (and nucleotide sequences coding for equivalent toxins) having the same or similar pesticidal activity of the exemplified toxin.
- Equivalent toxins will have amino acid homology with an exemplified toxin. This amino acid homology will typically be greater than 75%, preferably be greater than 90%, and most preferably be greater than 95%.
- amino acids may be placed in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. Below is a listing of examples of amino acids belonging to each class.
- non-conservative substitutions can also be made.
- the critical factor is that these substitutions must not significantly detract from the biological activity of the toxin.
- Recombinant hosts The genes encoding the toxins of the subject invention can be introduced into a wide variety of microbial or plant hosts. Expression of the toxin gene results, directly or indirectly, in the intracellular production and maintenance of the pesticide. Conjugal transfer and recombinant transfer can be used to create a Bt strain that expresses both toxins of the subject invention. Other host organisms may also be transformed with one or both of the toxin genes then used to accomplish the synergistic effect. With suitable microbial hosts, e.g., Pseudomonas, the microbes can be applied to the situs of the pest, where they will proliferate and be ingested. The result is control of the pest. Alternatively, the microbe hosting the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which retains the toxic activity, then can be applied to the environment of the target pest.
- suitable microbial hosts e.g., Pseudomonas
- the Bt toxin gene is introduced via a suitable vector into a microbial host, and said host is applied to the environment in a living state, it is essential that certain host microbes be used.
- Microorganism hosts are selected which are known to occupy the "phytosphere" (phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops of interest. These microorganisms are selected so as to be capable of successfully competing in the particular environment (crop and other insect habitats) with the wild-type microorganisms, provide for stable maintenance and expression of the gene expressing the polypeptide pesticide, and, desirably, provide for improved protection of the pesticide from environmental degradation and inactivation.
- microorganisms are known to inhabit the phylloplane (the surface of the plant leaves) and/or the rhizosphere (the soil surrounding plant roots) of a wide variety of important crops. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, e.g., genera Pseudomonas, Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas,
- yeast e.g., genera Saccharomyces, Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium.
- phytosphere bacterial species are Pseudomonas syringae,
- Pseudomonas fluorescens Serratia marcescens, Acetobacter xylinum, Agrobactenium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotobacter vinlandii; and phytosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.
- Rhodotorula rubra R. glutinis, R. marina, R. auranti
- Bacillus thuringiensis or recombinant cells expressing the Bt toxins can be treated to prolong the toxin activity and stabilize the cell.
- the pesticide microcapsule that is formed comprises the Bt toxin or toxins within a cellular structure that has been stabilized and will protect the toxin when the microcapsule is applied to the environment of the target pest.
- Suitable host cells may include either prokaryotes or eukaryotes, normally being limited to those cells which do not produce substances toxic to higher organisms, such as mammals. However, organisms which produce substances toxic to higher organisms could be used, where the toxic substances are unstable or the level of application sufficiently low as to avoid any possibility of toxicity to a mammalian host.
- hosts of particular interest will be the prokaryotes and the lower eukaryotes, such as fungi.
- the cell will usually be intact and be substantially in the proliferative form when treated, rather than in a spore form, although in some instances spores may be employed.
- Treatment of the microbial cell can be by chemical or physical means, or by a combination of chemical and/or physical means, so long as the technique does not deleteriously affect the properties of the toxin, nor diminish the cellular capability of protecting the toxin.
- chemical reagents are halogenating agents, particularly halogens of atomic no. 17-80. More particularly, iodine can be used under mild conditions and for sufficient time to achieve the desired results.
- aldehydes such as glutaraldehyde
- anti-infectives such as zephiran chloride and cetylpyridinium chloride
- alcohols such as isopropyl and ethanol
- histologic fixatives such as Lugol iodine, Bouin's fixative, various acids and Helly's fixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H. Freeman and Company, 1967); or a combination of physical (heat) and chemical agents that preserve and prolong the activity of the toxin produced in the cell when the cell is administered to the host environment.
- Examples of physical means are short wavelength radiation such as gamma-radiation and X-radiation, freezing, UV irradiation, lyophilization, and the like.
- Methods for treatment of microbial cells are disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which are incorporated herein by reference.
- the cells generally will have enhanced structural stability which will enhance resistance to environmental conditions.
- the method of cell treatment should be selected so as not to inhibit processing of the proform to the mature form of the pesticide by the target pest pathogen.
- formaldehyde will crosslink proteins and could inhibit processing of the proform of a polypeptide pesticide.
- the method of treatment should retain at least a substantial portion of the bio-availability or bioactivity of the toxin.
- Characteristics of particular interest in selecting a host cell for purposes of production include ease of introducing the B.t. gene or genes into the host, availability of expression systems, efficiency of expression, stability of the pesticide in the host, and the presence of auxiliary genetic capabilities.
- Characteristics of interest for use as a pesticide microcapsule include protective qualities for the pesticide, such as thick cell walls, pigmentation, and intracellular packaging or formation of inclusion bodies; survival in aqueous environments; lack of mammalian toxicity; attractiveness to pests for ingestion; ease of killing and fixing without damage to the toxin; and the like. Other considerations include ease of formulation and handling, economics, storage stability, and the like.
- the cellular host containing the B.t. insecticidal gene or genes may be grown in any convenient nutrient medium, where the DNA construct provides a selective advantage, providing for a selective medium so that substantially all or all of the cells retain the B.t. gene. These cells may then be harvested in accordance with conventional ways. Alternatively, the cells can be treated prior to harvesting.
- the B.t. cells producing the toxins of the invention can be cultured using standard art media and fermentation techniques. Upon completion of the fermentation cycle the bacteria can be harvested by first separating the B.t. spores and crystals from the fermentation broth by means well known in the art. The recovered B.t. spores and crystals can be formulated into a wettable powder, liquid concentrate, granules or other formulations by the addition of surfactants, dispersants, inert carriers, and other components to facilitate handling and application for particular target pests. These formulations and application procedures are all well known in the art.
- Formulated bait granules containing an attractant and spores, crystals, and toxins of the B.t. isolates, or recombinant microbes comprising the genes obtainable from the B.t. isolates disclosed herein can be applied to the soil.
- Formulated product can also be applied as a seed-coating or root treatment or total plant treatment at later stages of the crop cycle. Plant and soil treatments of B.t.
- cells may be employed as wettable powders, granules or dusts, by mixing with various inert materials, such as inorganic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (powdered corncobs, rice hulls, walnut shells, and the like).
- the formulations may include spreader-sticker adjuvants, stabilizing agents, other pesticidal additives, or surfactants.
- Liquid formulations may be aqueous-based or non-aqueous and employed as foams, gels, suspensions, emulsifiable concentrates, or the like.
- the ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.
- the pesticidal concentration will vary widely depending upon the nature of the particular formulation, particularly whether it is a concentrate or to be used directly.
- the pesticide will be present in at least 1% by weight and may be 100% by weight.
- the dry formulations will have from about 1-95% by weight of the pesticide while the liquid formulations will generally be from about 1 -60% by weight of the solids in the liquid phase.
- the formulations will generally have from about 10 2 to about 10 4 cells/mg. These formulations will be administered at about 50 mg (liquid or dry) to 1 kg or more per hectare.
- the formulations can be applied to the environment of the lepidopteran pest, e.g., foliage or soil, by spraying, dusting, sprinkling, or the like.
- a preferred recombinant host for production of the insecticidal proteins of the subject invention is a transformed plant.
- Genes encoding Bt toxin proteins, as disclosed herein, can be inserted into plant cells using a variety of techniques which are well known in the art. For example, a large number of cloning vectors comprising a replication system in Escherichia coli and a marker that permits selection of the transformed cells are available for preparation for the insertion of foreign genes into higher plants.
- the vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, inter alia.
- the DNA fragment having the sequence encoding the Bt toxin protein can be inserted into the vector at a suitable restriction site.
- the resulting plasmid is used for transformation into E. coli.
- the E. coli cells are cultivated in a suitable nutrient medium, then harvested and lysed.
- the plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular biological methods are generally carried out as methods of analysis.
- the DNA sequence used can be cleaved and joined to the next DNA sequence.
- Each plasmid sequence can be cloned in the same or other plasmids. Depending on the method of inserting desired genes into the plant, other DNA sequences may be necessary.
- the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right border, but often the right and the left border of the Ti or Ri plasmid T-DNA, has to be joined as the flanking region of the genes to be inserted.
- T-DNA for the transformation of plant cells has been intensively researched and sufficiently described in EP 120 516, Lee and Gelvin (2008), Hoekema (1985), Fraley et ah, (1986), and An et ah, (1985), and is well established in the art.
- the transformation vector normally contains a selectable marker that confers on the transformed plant cells resistance to a biocide or an antibiotic, such as Bialaphos,
- Kanamycin, G418, Bleomycin, or Hygromycin inter alia.
- the individually employed marker should accordingly permit the selection of transformed cells rather than cells that do not contain the inserted DNA.
- a large number of techniques are available for inserting DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agent, fusion, injection, biolistics
- the DNA to be inserted has to be cloned into special plasmids, namely either into an intermediate vector or into a binary vector.
- the intermediate vectors can be integrated into the Ti or Ri plasmid by homologous
- the Ti or Ri plasmid also comprises the vir region necessary for the transfer of the T-DNA.
- intermediate vectors cannot replicate themselves in Agrobacteria.
- the intermediate vector can be transferred into Agrobacterium tumefaciens by means of a helper plasmid
- Binary vectors can replicate themselves both in E. coli and in Agrobacteria. They comprise a selection marker gene and a linker or polylinker which are framed by the Right and Left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et ah, 1978).
- the Agrobacterium used as host cell is to comprise a plasmid carrying a vir region. The vir region is necessary for the transfer of the T-DNA into the plant cell. Additional T-DNA may be contained. The bacterium so transformed is used for the transformation of plant cells.
- Plant explants can advantageously be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes for the transfer of the DNA into the plant cell.
- Whole plants can then be regenerated from the infected plant material (for example, pieces of leaf, segments of stalk, roots, but also protoplasts or suspension- cultivated cells) in a suitable medium, which may contain antibiotics or biocides for selection.
- the plants so obtained can then be tested for the presence of the inserted DNA.
- No special demands are made of the plasmids in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, pUC derivatives.
- the transformed cells grow inside the plants in the usual manner. They can form germ cells and transmit the transformed trait(s) to progeny plants. Such plants can be grown in the normal manner and crossed with plants that have the same transformed hereditary factors or other hereditary factors. The resulting hybrid individuals have the corresponding phenotypic properties.
- plants will be transformed with genes wherein the codon usage has been optimized for plants. See, for example, U.S. Patent No. 5,380,831, which is hereby incorporated by reference. While some truncated toxins are exemplified herein, it is well-known in the Bt art that 130 kDa-type (full-length) toxins have an N-terminal half that is the core toxin, and a C-terminal half that is the protoxin "tail.” Thus, appropriate "tails" can be used with truncated / core toxins of the subject invention. See e.g. U.S. Patent No. 6,218,188 and U.S. Patent No. 6,673,990.
- a preferred transformed plant is a fertile maize plant comprising a plant expressible gene encoding a Vip3Ab protein, and further comprising a second plant expressible gene encoding a CrylCa protein.
- Transfer (or introgression) of the Vip3Ab - and CrylCa-determined trait(s) into inbred maize lines can be achieved by recurrent selection breeding, for example by backcrossing.
- a desired recurrent parent is first crossed to a donor inbred (the non-recurrent parent) that carries the appropriate gene(s) for the Vip3Ab - and Cry 1 (redetermined traits.
- the progeny of this cross is then mated back to the recurrent parent followed by selection in the resultant progeny for the desired trait(s) to be transferred from the non-recurrent parent.
- the progeny will be heterozygous for loci controlling the trait(s) being transferred, but will be like the recurrent parent for most or almost all other genes (see, for example, Poehlman & Sleper (1995) Breeding Field Crops, 4th Ed., 172-175; Fehr (1987) Principles of Cultivar Development, Vol. 1 : Theory and Technique, 360-376).
- IRM Insect Resistance Management
- Structured refuges 20% non-Lepidopteran Bt corn refuge in Corn Belt;
- Strips must be at least 4 rows wide (preferably 6 rows) to reduce
- the refuge strips can be planted as strips within the Bt field; the refuge strips must be at least 4 rows wide
- the above percentages, or similar refuge ratios, can be used for the subject double or triple stacks or pyramids.
- a goal would be zero refuge (or less than 5% refuge, for example). This is particularly true for commercial acreage - of over 10 acres for example.
- Example 1- Production and trypsin processing of Vip3Ab and CrylCa proteins.
- the genes encoding the CrylCa and Vip3Abl pro toxins were expressed in Pseudomonas fluorescens expression strains and the full length proteins isolated as insoluble inclusion bodies.
- the washed inclusion bodies were solubilized by stirring at 37 °C in buffer containing 20 mM CAPS buffer, pH 1 1, + 10 mM DDT, + 0.1% 2- mercaptoethanol, for 2 hrs.
- the solution was centrifuged at 27,000 x g for 10 min. at 37 °C and the supernatant treated with 0.5% (w/v) TCPK treated trypsin (Sigma). This solution was incubated with mixing for an additional 1 hr.
- the combined fractions of the purified toxin were concentrated and loaded onto a Superose 6 column (1.6 cm dia., 60 cm long), and further purified by size exclusion chromatography. Fractions comprising a single peak of the monomeric molecular weight were combined, and concentrated, resulting in a preparation more than 95% homogeneous for a protein having a molecular weight of about 60,000 kDa.
- Vip3Abl Processing of Vip3Abl was achieved in a similar manner starting with the purified full length 85 kDa protein (DIG-307).
- the protein (12 mg) was dialyzed into 50 mM sodium phosphate buffer, pH 8.4, then processed by adding 1 mg of solid trypsin and incubating for 1 hrs. at room temperature.
- the solution was loaded onto a MonoQ anion exchange column (1 cm dia., 10 cm. long), and eluted with a linear gradient of NaCl from 0 to 500 mM in 20 mM sodium phosphate buffer, pH 8.4 over 7 column volumes. Elution of the protein was monitored by SDS-PAGE.
- the major processed band had a molecular weight of 65 kDa, as determined by SDS-PAGE using molecular weight standards for comparison.
- Example 2 Iodination of CrylCa core toxin protein
- the CrylCa core toxin segment (residues 29-619) contains two cysteine amino acid residues, at positions 210 and 438.
- Palmer et al. (1997) demonstrated that the phenyl rings of fluorescein-5- maleimide can be radio-iodinated and then reacted with proteins that contain sulfhydryl groups (e.g. as provided by free cysteine residues), resulting in alkylation of the free cysteines in the protein, and thus providing a radioactively labeled protein.
- the trypsin- truncated CrylCa core toxin contains two cysteine residues and thus provides a substrate for alkylation and radiolabeling of the protein at these two (specific) sites.
- Fluorescein-5-maleimide was dissolved to 10 mM in DMSO (Dimethyl Sulfoxide), then diluted to 1 mM in phosphate buffered saline (PBS; 20 mM sodium phosphate, 0.15 M NaCl, pH7.5), as determined by the molar extinction coefficient of F 5- M (68,000 M ⁇ cm "1 ).
- PBS phosphate buffered saline
- F 5- M phosphate buffered saline
- the solution was removed from the iodination reaction by pipetting and 2 ⁇ g of highly purified trypsin-truncated CrylCa core toxin protein in PBS were added to the solution.
- the protein was incubated at 4° with the iodinated F 5-M solution for 48 hrs, when the reaction was terminated by adding ⁇ - mercaptoethanol to 14 mM final concentration.
- the reaction mixture was added to a ZebraTM spin column (Invitrogen) equilibrated in 20 mM CAPS, 150 mM KC1, pH9, and centrifuged at 1500 x g for 2 min to separate non-reacted iodinated dye from the protein.
- the 125 I radiolabeled fluorescein-CrylCa core toxin protein was counted in a gamma counter to determine its specific radioactivity, assuming 80% recovery of the input toxin protein.
- the specific activity of the radiolabeled CrylCa core toxin protein was approximately 6.8 ⁇ / ⁇ protein.
- the radiolabeled protein was also characterized by SDS- PAGE and visualized by phosphor-imaging to validate that the radioactivity measured was covalently associated with the CrylCa core toxin protein.
- Coomassie stained SDS-PAGE gels were imaged by wrapping them in MylarTM film (12 ⁇ thick), and exposing them under a Molecular Dynamics (Sunnyvale, CA) storage phosphor screen (35 cm x 43 cm) for 1 hour. The plates were developed using a Molecular Dynamics Storm 820 phosphor- imager and the image analyzed using ImageQuantTM software.
- radioactivity was detectable in the gel region well below the CrylCa core toxin protein band (i.e. fragments smaller than the CrylCa core toxin protein at about 10 kDa in size and lower). These radioactive contaminants likely represent small peptides probably associated in the truncated CrylCa protein due to the action of the trypsin used to cleave the protein to its core structure.
- Example 3 Competitive binding assays to BBMVs from S. frugiperda with core toxin proteins of CrylCa and Vip3Ab.
- Concentrations of the homologous competitive non-radiolabeled CrylCa core toxin protein added to the reaction mixture was 0.1, 1, 10, 100, and 1000 nM.
- the heterologous trypsin truncated Vip3Ab protein was tested at 10 and 1,000 nM and the proteins were added at the same time as the radioactive CrylCa core toxin protein to assure true binding competition.
- the bottom the centrifuge tube was cut out and the protein pellet contained within this section placed in a 13 x 100 mm glass culture tube and counted in a gamma counter for 10 minutes to obtain the amount of bound radioactivity contained the pellet fraction.
- the amount of radioactivity in the bound protein fraction provides an indication of the amount of Cry protein bound to the insect receptor
- Non-specific binding was represented by the counts obtained in the pellet in the presence of 1,000 nM of non-radiolabeled CrylCa core toxin protein.
- the amount of radiolabeled CrylCa specifically bound to the BBMV (specific binding) was measured by subtracting the level of total binding from non specific binding.
- One hundred percent total binding was considered to be the amount of binding in the absence of any competitor Cry 1 Fa core toxin protein. The data is expressed as percent of specific bound 125 I CrylCa versus concentration of competitive unlabeled ligand.
- Figure 1 is a dose response curve for the displacement of 125 I radiolabeled fluorescein-5-maleimide trypsin-truncated CrylCa in BBMV's from S. frugiperda (FAW) larvae.
- the figure shows the ability of non-labled CrylCa ( ⁇ ) to displace the labeled CrylCa in a dose dependent manner in the range from 0.1 to 1,000 nM.
- the chart plots the percent of specifically bound labeled CrylCa (total bound minus non-specific bound) versus the concentration of the non-radiolabeled ligands added.
- the inability of non radiolabeled Vip3Abl (A) at 10 and 1,000 nM to displace the specifically bound radiolabeled CrylCa is shown.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Dentistry (AREA)
- Agronomy & Crop Science (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Insects & Arthropods (AREA)
- Botany (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Virology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Catching Or Destruction (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SI201031566T SI2512225T1 (en) | 2009-12-16 | 2010-12-16 | Use of vip3ab in combination with cry1ca for management of resistant insects |
RS20171046A RS56508B1 (en) | 2009-12-16 | 2010-12-16 | Use of vip3ab in combination with cry1ca for management of resistant insects |
PL10842622T PL2512225T3 (en) | 2009-12-16 | 2010-12-16 | Use of vip3ab in combination with cry1ca for management of resistant insects |
HRP20171475TT HRP20171475T1 (en) | 2009-12-16 | 2017-10-03 | Use of vip3ab in combination with cry1ca for management of resistant insects |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US28428109P | 2009-12-16 | 2009-12-16 | |
US28427509P | 2009-12-16 | 2009-12-16 | |
PCT/US2010/060835 WO2011084634A1 (en) | 2009-12-16 | 2010-12-16 | Use of vip3ab in combination with cry1ca for management of resistant insects |
Publications (3)
Publication Number | Publication Date |
---|---|
EP2512225A1 true EP2512225A1 (en) | 2012-10-24 |
EP2512225A4 EP2512225A4 (en) | 2013-06-05 |
EP2512225B1 EP2512225B1 (en) | 2017-08-23 |
Family
ID=44305723
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10842622.2A Active EP2512225B1 (en) | 2009-12-16 | 2010-12-16 | Use of vip3ab in combination with cry1ca for management of resistant insects |
Country Status (24)
Country | Link |
---|---|
US (1) | US20130203657A1 (en) |
EP (1) | EP2512225B1 (en) |
JP (1) | JP5908843B2 (en) |
KR (1) | KR101841293B1 (en) |
CN (1) | CN102753013B (en) |
AR (1) | AR079624A1 (en) |
AU (1) | AU2010339833B2 (en) |
BR (1) | BR112012014803B1 (en) |
CA (1) | CA2782576A1 (en) |
CL (1) | CL2012001628A1 (en) |
CO (1) | CO6561803A2 (en) |
DK (1) | DK2512225T3 (en) |
ES (1) | ES2641493T3 (en) |
HR (1) | HRP20171475T1 (en) |
HU (1) | HUE034006T2 (en) |
IL (1) | IL220342A (en) |
MX (1) | MX348226B (en) |
NZ (1) | NZ601099A (en) |
PL (1) | PL2512225T3 (en) |
PT (1) | PT2512225T (en) |
RS (1) | RS56508B1 (en) |
SI (1) | SI2512225T1 (en) |
WO (1) | WO2011084634A1 (en) |
ZA (1) | ZA201204922B (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2782557C (en) | 2009-12-16 | 2023-02-28 | Dow Agrosciences Llc | Use of cry1da in combination with cry1ca for management of resistant insects |
CN112400900A (en) | 2012-03-09 | 2021-02-26 | 韦斯塔隆公司 | Combination of toxic peptide production, peptide expression in plants and cysteine-rich peptides |
US11692016B2 (en) | 2012-03-09 | 2023-07-04 | Vestaron Corporation | High gene expression yeast strain |
CA2886787A1 (en) * | 2012-10-05 | 2014-04-10 | Dow Agrosciences Llc | Use of cry1ea in combinations for management of resistant fall armyworm insects |
JP6396330B2 (en) * | 2013-02-12 | 2018-09-26 | フラウンホーファ−ゲゼルシャフト ツァー フォルデルング デア アンゲバンデン フォルシュンク エー. ファオ.Fraunhofer−Gesellschaft Zur Forderung Der Angewandten Forschung E. V. | Polypeptides against phytopathogenic fungi |
CN104292314B (en) * | 2013-07-19 | 2017-11-17 | 中国科学院亚热带农业生态研究所 | The Cry1Ca of codon optimization#The method of gene and recombinant vector and change crop resistance |
CN103570810B (en) * | 2013-09-27 | 2016-10-05 | 杭州师范大学 | A kind of bacillus thuringiensis Vegetative Insecticidal Proteins, gene, carrier and application |
CN109152347A (en) * | 2016-04-19 | 2019-01-04 | 美国陶氏益农公司 | For managing the combination of four kinds of VIP and CRY proteotoxins of the insect pest in plant |
CN110267527A (en) | 2016-10-21 | 2019-09-20 | 韦斯塔隆公司 | Cleavable peptide and insecticidal and nematicidal albumen comprising cleavable peptide |
US11129906B1 (en) | 2016-12-07 | 2021-09-28 | David Gordon Bermudes | Chimeric protein toxins for expression by therapeutic bacteria |
RU2020114356A (en) | 2017-09-26 | 2021-10-27 | ДАУ АГРОСАЙЕНСИЗ ЭлЭлСи | CHIMERIC INSECTICIDE PROTEINS |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0400246A1 (en) * | 1989-05-31 | 1990-12-05 | Plant Genetic Systems, N.V. | Prevention of Bt resistance development |
US5877012A (en) * | 1993-03-25 | 1999-03-02 | Novartis Finance Corporation | Class of proteins for the control of plant pests |
US7129212B2 (en) * | 1996-10-30 | 2006-10-31 | Mycogen Corporation | Polynucleotides, pesticidal proteins, and novel methods of using them |
US6603063B1 (en) * | 1999-05-07 | 2003-08-05 | Mycogen Corp. | Plants and cells transformed with a nucleic acid from Bacillus thuringiensis strain KB59A4-6 encoding a novel SUP toxin |
US5942664A (en) * | 1996-11-27 | 1999-08-24 | Ecogen, Inc. | Bacillus thuringiensis Cry1C compositions toxic to lepidopteran insects and methods for making Cry1C mutants |
US6750379B2 (en) * | 2000-03-09 | 2004-06-15 | Dekalb Genetics Corporation | Homologous recombination-mediated transgene alterations in plants |
US7355099B2 (en) * | 2003-02-20 | 2008-04-08 | Athenix Corporation | AXMI-004, a delta-endotoxin gene and methods for its use |
AR048747A1 (en) * | 2004-03-05 | 2006-05-24 | Agrigenetics Inc | COMBINATIONS OF CRY1AB AND CRY1FA AS A TOOL FOR CONTROL OF INSECT RESISTANCE |
CA2621874C (en) * | 2005-09-08 | 2014-12-16 | Chromatin Inc. | Plants modified with mini-chromosomes |
US9994621B2 (en) * | 2007-06-01 | 2018-06-12 | Bayer Cropscience N.V. | Genes encoding insecticidal proteins |
WO2009003130A2 (en) * | 2007-06-26 | 2008-12-31 | Reactive Nanotechnologies, Inc. | Gasketless low-temperature hermetic sealing with solder |
MX2010011925A (en) * | 2008-05-01 | 2010-11-30 | Bayer Bioscience Nv | Armyworm insect resistance management in transgenic plants. |
CA2758885C (en) * | 2009-04-17 | 2018-09-11 | Dow Agrosciences Llc | Dig-3 insecticidal cry toxins |
CN103739683B (en) * | 2014-01-17 | 2015-06-17 | 北京大北农科技集团股份有限公司 | Insecticidal protein, and encoding gene and use thereof |
-
2010
- 2010-12-16 CA CA2782576A patent/CA2782576A1/en not_active Abandoned
- 2010-12-16 KR KR1020127018259A patent/KR101841293B1/en active IP Right Grant
- 2010-12-16 PT PT108426222T patent/PT2512225T/en unknown
- 2010-12-16 RS RS20171046A patent/RS56508B1/en unknown
- 2010-12-16 US US13/516,642 patent/US20130203657A1/en not_active Abandoned
- 2010-12-16 PL PL10842622T patent/PL2512225T3/en unknown
- 2010-12-16 EP EP10842622.2A patent/EP2512225B1/en active Active
- 2010-12-16 CN CN201080063907.2A patent/CN102753013B/en active Active
- 2010-12-16 HU HUE10842622A patent/HUE034006T2/en unknown
- 2010-12-16 AU AU2010339833A patent/AU2010339833B2/en not_active Ceased
- 2010-12-16 SI SI201031566T patent/SI2512225T1/en unknown
- 2010-12-16 ES ES10842622.2T patent/ES2641493T3/en active Active
- 2010-12-16 WO PCT/US2010/060835 patent/WO2011084634A1/en active Application Filing
- 2010-12-16 DK DK10842622.2T patent/DK2512225T3/en active
- 2010-12-16 BR BR112012014803-6A patent/BR112012014803B1/en active IP Right Grant
- 2010-12-16 MX MX2012007122A patent/MX348226B/en active IP Right Grant
- 2010-12-16 JP JP2012544849A patent/JP5908843B2/en active Active
- 2010-12-16 AR ARP100104684 patent/AR079624A1/en not_active Application Discontinuation
- 2010-12-16 NZ NZ60109910A patent/NZ601099A/en not_active IP Right Cessation
-
2012
- 2012-06-12 IL IL220342A patent/IL220342A/en not_active IP Right Cessation
- 2012-06-15 CL CL2012001628A patent/CL2012001628A1/en unknown
- 2012-07-02 ZA ZA2012/04922A patent/ZA201204922B/en unknown
- 2012-07-16 CO CO12119350A patent/CO6561803A2/en not_active Application Discontinuation
-
2017
- 2017-10-03 HR HRP20171475TT patent/HRP20171475T1/en unknown
Non-Patent Citations (5)
Title |
---|
, 1 December 2009 (2009-12-01), XP55060013, Retrieved from the Internet: URL:http://combat-monsanto.org/IMG/pdf/btcrops.pdf [retrieved on 2013-04-17] * |
, 1 January 2009 (2009-01-01), XP55060016, Retrieved from the Internet: URL:http://www.agry.purdue.edu/CCA/2008/Proceedings/Steffey.pdf [retrieved on 2013-04-17] * |
, 9 December 2008 (2008-12-09), XP55060010, DOI: 10.1094/PHP-2004- Retrieved from the Internet: URL:http://www.pnas.org/content/105/49/19029.full.pdf#page=1&view=FitH [retrieved on 2013-04-17] * |
PITTENDRIGH B R ET AL: "''Active'' refuges can inhibit the evolution of resistance in insects towards transgenic insect-resistant plants", JOURNAL OF THEORETICAL BIOLOGY, ACADEMIC PRESS, LONDON, GB, vol. 231, no. 4, 21 December 2004 (2004-12-21), pages 461-474, XP004635702, ISSN: 0022-5193, DOI: 10.1016/J.JTBI.2004.05.023 * |
See also references of WO2011084634A1 * |
Also Published As
Publication number | Publication date |
---|---|
NZ601099A (en) | 2014-10-31 |
MX348226B (en) | 2017-05-29 |
PL2512225T3 (en) | 2017-12-29 |
IL220342A0 (en) | 2012-08-30 |
RU2012129912A (en) | 2014-01-27 |
ES2641493T3 (en) | 2017-11-10 |
KR101841293B1 (en) | 2018-03-22 |
MX2012007122A (en) | 2015-05-11 |
ZA201204922B (en) | 2013-03-27 |
WO2011084634A1 (en) | 2011-07-14 |
HUE034006T2 (en) | 2018-01-29 |
CA2782576A1 (en) | 2011-07-14 |
RS56508B1 (en) | 2018-02-28 |
HRP20171475T1 (en) | 2017-12-29 |
US20130203657A1 (en) | 2013-08-08 |
DK2512225T3 (en) | 2017-11-06 |
AU2010339833A1 (en) | 2012-07-12 |
EP2512225B1 (en) | 2017-08-23 |
BR112012014803A2 (en) | 2015-08-25 |
BR112012014803B1 (en) | 2024-01-30 |
CN102753013A (en) | 2012-10-24 |
AR079624A1 (en) | 2012-02-08 |
JP5908843B2 (en) | 2016-04-26 |
CN102753013B (en) | 2015-10-07 |
KR20120115979A (en) | 2012-10-19 |
EP2512225A4 (en) | 2013-06-05 |
IL220342A (en) | 2016-07-31 |
AU2010339833B2 (en) | 2016-03-31 |
JP2013514777A (en) | 2013-05-02 |
PT2512225T (en) | 2017-11-10 |
SI2512225T1 (en) | 2017-12-29 |
CO6561803A2 (en) | 2012-11-15 |
CL2012001628A1 (en) | 2013-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2513317B1 (en) | Use of cry1da in combination with cry1be for management of resistant insects | |
US9663795B2 (en) | Use of Cry1Ab in combination with Cry1Be for management of resistant insects | |
EP2512219B1 (en) | Combined use of vip3ab and cry1fa for management of resistant insects | |
AU2010330917B2 (en) | Combined use of Cry1Ca and Cry1Fa proteins for insect resistance management | |
EP2512225B1 (en) | Use of vip3ab in combination with cry1ca for management of resistant insects | |
EP2513316B1 (en) | Use of cry1da in combination with cry1ca for management of resistant insects | |
US9045766B2 (en) | Combined use of Vip3Ab and Cry1Ab for management of resistant insects | |
EP2739133A1 (en) | Use of dig3 insecticidal crystal protein in combination with cry1ab | |
US10119149B2 (en) | Use of DIG3 insecticidal crystal protein in combination with cry1Ab for management of resistance in european cornborer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20120704 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: ME |
|
RAX | Requested extension states of the european patent have changed |
Extension state: ME Payment date: 20120704 |
|
A4 | Supplementary search report drawn up and despatched |
Effective date: 20130507 |
|
RIC1 | Information provided on ipc code assigned before grant |
Ipc: C07K 14/325 20060101ALI20130429BHEP Ipc: A01H 5/00 20060101AFI20130429BHEP Ipc: C12N 15/82 20060101ALI20130429BHEP |
|
17Q | First examination report despatched |
Effective date: 20140710 |
|
GRAP | Despatch of communication of intention to grant a patent |
Free format text: ORIGINAL CODE: EPIDOSNIGR1 |
|
INTG | Intention to grant announced |
Effective date: 20170118 |
|
GRAJ | Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted |
Free format text: ORIGINAL CODE: EPIDOSDIGR1 |
|
INTC | Intention to grant announced (deleted) | ||
GRAR | Information related to intention to grant a patent recorded |
Free format text: ORIGINAL CODE: EPIDOSNIGR71 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAJ | Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted |
Free format text: ORIGINAL CODE: EPIDOSDIGR1 |
|
GRAR | Information related to intention to grant a patent recorded |
Free format text: ORIGINAL CODE: EPIDOSNIGR71 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAJ | Information related to disapproval of communication of intention to grant by the applicant or resumption of examination proceedings by the epo deleted |
Free format text: ORIGINAL CODE: EPIDOSDIGR1 |
|
GRAR | Information related to intention to grant a patent recorded |
Free format text: ORIGINAL CODE: EPIDOSNIGR71 |
|
GRAS | Grant fee paid |
Free format text: ORIGINAL CODE: EPIDOSNIGR3 |
|
GRAA | (expected) grant |
Free format text: ORIGINAL CODE: 0009210 |
|
INTG | Intention to grant announced |
Effective date: 20170706 |
|
INTC | Intention to grant announced (deleted) | ||
AK | Designated contracting states |
Kind code of ref document: B1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
AX | Request for extension of the european patent |
Extension state: ME |
|
INTG | Intention to grant announced |
Effective date: 20170717 |
|
REG | Reference to a national code |
Ref country code: GB Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: EP |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: REF Ref document number: 920290 Country of ref document: AT Kind code of ref document: T Effective date: 20170915 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: FG4D |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: TUEP Ref document number: P20171475 Country of ref document: HR |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R096 Ref document number: 602010044731 Country of ref document: DE |
|
REG | Reference to a national code |
Ref country code: CH Ref legal event code: NV Representative=s name: FIAMMENGHI-FIAMMENGHI, CH |
|
REG | Reference to a national code |
Ref country code: RO Ref legal event code: EPE |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: FP |
|
REG | Reference to a national code |
Ref country code: DK Ref legal event code: T3 Effective date: 20171102 |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: FG2A Ref document number: 2641493 Country of ref document: ES Kind code of ref document: T3 Effective date: 20171110 Ref country code: PT Ref legal event code: SC4A Ref document number: 2512225 Country of ref document: PT Date of ref document: 20171110 Kind code of ref document: T Free format text: AVAILABILITY OF NATIONAL TRANSLATION Effective date: 20171026 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 8 |
|
REG | Reference to a national code |
Ref country code: EE Ref legal event code: FG4A Ref document number: E014352 Country of ref document: EE Effective date: 20171018 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: T1PR Ref document number: P20171475 Country of ref document: HR |
|
REG | Reference to a national code |
Ref country code: LT Ref legal event code: MG4D |
|
REG | Reference to a national code |
Ref country code: HU Ref legal event code: AG4A Ref document number: E034006 Country of ref document: HU |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: NO Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171123 Ref country code: SE Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 Ref country code: FI Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 Ref country code: LT Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IS Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171223 Ref country code: LV Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 Ref country code: GR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20171124 |
|
REG | Reference to a national code |
Ref country code: SK Ref legal event code: T3 Ref document number: E 25800 Country of ref document: SK |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R097 Ref document number: 602010044731 Country of ref document: DE |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: SM Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
PLBE | No opposition filed within time limit |
Free format text: ORIGINAL CODE: 0009261 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT |
|
26N | No opposition filed |
Effective date: 20180524 |
|
REG | Reference to a national code |
Ref country code: IE Ref legal event code: MM4A |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: LU Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20171216 Ref country code: MT Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20171216 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: IE Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20171216 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: ODRP Ref document number: P20171475 Country of ref document: HR Payment date: 20181102 Year of fee payment: 9 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MC Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CY Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20170823 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R082 Ref document number: 602010044731 Country of ref document: DE Representative=s name: ZWICKER SCHNAPPAUF & PARTNER PATENTANWAELTE PA, DE |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: MK Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: ODRP Ref document number: P20171475 Country of ref document: HR Payment date: 20191114 Year of fee payment: 10 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: TR Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: AL Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT Effective date: 20170823 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: ODRP Ref document number: P20171475 Country of ref document: HR Payment date: 20201210 Year of fee payment: 11 |
|
REG | Reference to a national code |
Ref country code: AT Ref legal event code: UEP Ref document number: 920290 Country of ref document: AT Kind code of ref document: T Effective date: 20170823 |
|
REG | Reference to a national code |
Ref country code: DE Ref legal event code: R081 Ref document number: 602010044731 Country of ref document: DE Owner name: CORTEVA AGRISCIENCE LLC, INDIANAPOLIS, US Free format text: FORMER OWNER: DOW AGROSCIENCES LLC, INDIANAPOLIS, IND., US |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: ODRP Ref document number: P20171475 Country of ref document: HR Payment date: 20211216 Year of fee payment: 12 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: PNAN Ref document number: P20171475 Country of ref document: HR Owner name: CORTEVA AGRISCIENCE LLC, US |
|
REG | Reference to a national code |
Ref country code: NL Ref legal event code: HC Owner name: CORTEVA AGRISCIENCE LLC; US Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), CHANGE OF OWNER(S) NAME; FORMER OWNER NAME: DOW AGROSCIENCES, LLC Effective date: 20220127 |
|
REG | Reference to a national code |
Ref country code: BE Ref legal event code: HC Owner name: CORTEVA AGRISCIENCE LLC; US Free format text: DETAILS ASSIGNMENT: CHANGE OF OWNER(S), CHANGE OF OWNER(S) NAME; FORMER OWNER NAME: DOW AGROSCIENCES, LLC Effective date: 20211206 |
|
REG | Reference to a national code |
Ref country code: HU Ref legal event code: HC9C Owner name: CORTEVA AGRISCIENCE LLC, US Free format text: FORMER OWNER(S): DOW AGROSCIENCES, LLC, US |
|
REG | Reference to a national code |
Ref country code: EE Ref legal event code: HC1A Ref document number: E014352 Country of ref document: EE |
|
REG | Reference to a national code |
Ref country code: SK Ref legal event code: TC4A Ref document number: E 25800 Country of ref document: SK Owner name: CORTEVA AGRISCIENCE LLC, INDIANAPOLIS, IN 4626, US Effective date: 20220519 Ref country code: AT Ref legal event code: HC Ref document number: 920290 Country of ref document: AT Kind code of ref document: T Owner name: CORTEVA AGRISCIENCE LLC, US Effective date: 20220426 |
|
REG | Reference to a national code |
Ref country code: ES Ref legal event code: PC2A Owner name: CORTEVA AGRISCIENCE LLC Effective date: 20220830 |
|
REG | Reference to a national code |
Ref country code: SI Ref legal event code: SP73 Owner name: CORTEVA AGRISCIENCE LLC; US Effective date: 20220726 |
|
REG | Reference to a national code |
Ref country code: FR Ref legal event code: PLFP Year of fee payment: 13 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: ODRP Ref document number: P20171475 Country of ref document: HR Payment date: 20221213 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: SK Payment date: 20221115 Year of fee payment: 13 Ref country code: RS Payment date: 20221116 Year of fee payment: 13 Ref country code: RO Payment date: 20221122 Year of fee payment: 13 Ref country code: NL Payment date: 20221114 Year of fee payment: 13 Ref country code: IT Payment date: 20221111 Year of fee payment: 13 Ref country code: GB Payment date: 20221103 Year of fee payment: 13 Ref country code: EE Payment date: 20221103 Year of fee payment: 13 Ref country code: DK Payment date: 20221213 Year of fee payment: 13 Ref country code: CZ Payment date: 20221128 Year of fee payment: 13 Ref country code: BG Payment date: 20221116 Year of fee payment: 13 Ref country code: AT Payment date: 20221125 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: SI Payment date: 20221121 Year of fee payment: 13 Ref country code: PT Payment date: 20221216 Year of fee payment: 13 Ref country code: PL Payment date: 20221117 Year of fee payment: 13 Ref country code: HU Payment date: 20221126 Year of fee payment: 13 Ref country code: HR Payment date: 20221213 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: ES Payment date: 20230110 Year of fee payment: 13 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: FR Payment date: 20231226 Year of fee payment: 14 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: BE Payment date: 20231229 Year of fee payment: 14 |
|
PGFP | Annual fee paid to national office [announced via postgrant information from national office to epo] |
Ref country code: DE Payment date: 20231221 Year of fee payment: 14 Ref country code: CH Payment date: 20240102 Year of fee payment: 14 |
|
REG | Reference to a national code |
Ref country code: HR Ref legal event code: PBON Ref document number: P20171475 Country of ref document: HR Effective date: 20231216 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: RS Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20231216 |
|
REG | Reference to a national code |
Ref country code: DK Ref legal event code: EBP Effective date: 20231231 |
|
PG25 | Lapsed in a contracting state [announced via postgrant information from national office to epo] |
Ref country code: CZ Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES Effective date: 20231216 |